miRNeasy FFPE Kit

For purification of microRNA and total RNA from formalin-fixed, paraffin-embedded tissue sections

Effective purification of miRNA and total RNA

Novel method to overcome formalin crosslinking

Efficient release of RNA without compromising integrity

Streamlined protocol providing RNA in just 85 minutes

The miRNeasy FFPE Kit enables purification of total RNA that includes RNA from approximately 18 nucleotides upwards from formalin-fixed, paraffin-embedded (FFPE) tissue sections. The kit provides recovery of usable RNA fragments, including miRNA and other small RNAs, for applications such as quantitative, real-time RT-PCR. Procedures can be automated on theQIAcube.

Total RNA including miRNA was purified from the indicated rat tissues using either the miRNeasy FFPE Kit or phenol-chloroform extraction. Purified RNA was used as a template in quantitative, real-time RT-PCR assays for the miRNA miR-16 using the miScript PCR System. Results showed that for lung and liver tissues, CT values were lower after purification using the miRNeasy FFPE Kit, indicating that higher amounts of miRNA were purified than when using phenol-chloroform extraction. For kidney tissue, CT values were similar for both methods. CT values were similar or lower after purification using the miRNeasy FFPE Kit for all tissue types tested.|RNA was purified from FFPE sections of the indicated rat tissues using either the miRNeasy FFPE Kit or a similar kit from Supplier A. [A] RNA yields were determined by measuring absorbance at 260 nm. [B]Purified RNA was used as a template in quantitative, real-time RT-PCR assays for the miRNA miR-16 using the miScript PCR System. Results showed that CT values were lower after purification using the miRNeasy FFPE Kit, indicating that higher amounts of miRNA were purified than when using the kit from Supplier A.|Rat liver tissue was formalin fixed for 24 hours or 60 hours, followed by purification of total RNA including miRNA using the miRNeasy FFPE Kit. Purified RNA was used as a template in quantitative, real-time RT-PCR using the miScript PCR System to detect miRNAs miR-16 and miR-29a and for the larger mRNA of the PGK1 gene. Results show successful detection of both miRNAs as well as for the mRNA from the same eluate.||

Fixing tissues with formalin leads to RNA-RNA and RNA-protein crosslinking which impairs RNA performance in downstream applications. The miRNeasy FFPE Kit provides special lysis and incubation conditions to reverse formalin crosslinking of RNA. A specially developed lysis buffer efficiently releases RNA from tissue sections while avoiding further RNA degradation. The lysate is treated with DNase then optimized binding conditions allow purification of all usable RNA from approximately 18 nucleotides upwards.

Procedure

The entire miRNeasy FFPE procedure can be completed in as little as 85 minutes. The optimized lysis buffer allows sample lysis with proteinase K digestion in only 15 minutes without sacrificing RNA yields. After lysis, samples are incubated at 80ºC for 15 minutes to reverse formalin crosslinking. Genomic DNA is then rapidly removed in an optimized DNase step with DNase Booster Buffer, and concentrated RNA is purified using RNeasy MinElute spin columns (see flowchart "miRNeasy FFPE procedure"). Since RNA is eluted in a volume of just 14–30 µl, smaller reaction volumes are possible in downstream applications.

Applications

The miRNeasy FFPE Kit allows purification of miRNA with total RNA for use in a variety of applications, such as quantitative, real-time RT-PCR.

Feature

Specifications

Applications

PCR, qPRC, real-time RT-PCR, microArray

Elution volume

14-30 µl

Format

Spin column

Main sample type

FFPE tissue samples

Processing

Manual (protocol for automated processing on QIAcube available)

Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein

miRNA, total RNA

Sample amount

up to 4 sections, each with a thickness up to 10 µm and a surface area up to 250mm^2 (automated protocol on QIAcube: up to 2 sections)